Molecular stripping in the NFκB/IκB/DNA genetic regulatory network

Potoyan, Davit A.; Zheng, Weihua; Komives, Elizabeth A.; Wolynes, Peter G.

doi:10.1101/027912

Cited by 11 publications

(31 citation statements)

References 26 publications

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“…And the IKK (inhibitory κB (IκB) kinase)-mediated phosphorylation-induced proteasomal degradation of IκB inhibitor, enabling the active NF-κB to translocate to the nucleus (Vallabhapurapu and Karin, 2009). However, IκB could sequester NF-κB subunits and terminate the transcription activity (Potoyan et al, 2015). In our study, we confirmed the involvement of NF-κB pathway in the regulation by NMP on the hypoxia-mediated the reduction of osteoblast differentiation.…”

Section: Discussionsupporting

confidence: 85%

N-methyl pyrrolidone (NMP) ameliorates the hypoxia-reduced osteoblast differentiation via inhibiting the NF-κB signaling

Zhao

et al. 2016

J. Toxicol. Sci.

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-Ischemic-hypoxic condition for local osteoblasts and bone mesenchymal stem cells during bone fracture inhibits bone repairing. N-methyl pyrrolidone (NMP) has been approved as a safe and biologically inactive small chemical molecule, and might be useful for bone fracture repairing. In the present study, we investigated the effect of NMP on the hypoxia-reduced cellular viability and the expression of differentiation-associated markers, such as bone morphogenetic protein 2 (BMP-2), propeptide of type I procollagen I (PINP), alkaline phosphatase (ALP) or runt-related transcription factor 2 (Runx2) in the osteoblasts, and then we examined the molecular mechanism underlining such effect in the human osteoblastic hFOB 1.19 cells. Our results demonstrated that NMP significantly blocked the hypoxia-induced cell viability reduction and inhibited the hypoxia-caused expression downregulation of BMP-2, PINP, ALP and Runx2 in hFOB 1.19 cells. Then we confirmed the involvement of nuclear factor κB (NF-κB) pathway in the regulation by NMP on the hypoxia-mediated the reduction of osteoblast differentiation. The upregulated expression and transcriptional activity of NF-κB, while the downregulated inhibitory κB expression by the hypoxia treatment was reversed by the treatment with 10 mM NMP. In conclusion, our study found a protective role of NMP in osteoblast differentiation in response to hypoxia, and such protection was through inhibiting the NF-κB signaling. This suggests that NMP might be a protective agent in bone fracture repairing.

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Section: Discussionsupporting

confidence: 85%

N-methyl pyrrolidone (NMP) ameliorates the hypoxia-reduced osteoblast differentiation via inhibiting the NF-κB signaling

Zhao

et al. 2016

J. Toxicol. Sci.

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“…Successful competition by the PEST sequence should result in the dissociation of the NF-κB-IκBα complex from the DNA. Recent modeling studies (14) show that the PEST can adopt alternative conformations, both inside and outside the DNA binding cavity, allowing for the accommodation of the DNA in the ternary complex as well as for competition that results in the dissociation of the DNA.…”

Section: Discussionmentioning

confidence: 99%

Structural characterization of the ternary complex that mediates termination of NF-κB signaling by IκBα

Mukherjee

Quintas

McNulty

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The transcription factor NF-κB is used in many systems for the transduction of extracellular signals into the expression of signal-responsive genes. Published structural data explain the activation of NF-κB through degradation of its dedicated inhibitor IκBα, but the mechanism by which NF-κB-mediated signaling is turned off by its removal from the DNA in the presence of newly synthesized IκBα (termed stripping) is unknown. Previous kinetic studies showed that IκBα accelerates NF-κB dissociation from DNA, and a transient ternary complex between NF-κB, its cognate DNA sequence, and IκBα was observed. Here we structurally characterize the >100-kDa ternary complex by NMR and negative stain EM and show a modeled structure that is consistent with the measurements. These data provide a structural basis for previously unidentified insights into the molecular mechanism of stripping.transcriptional activation | NMR | negative stain electron microscopy | protein-DNA complex | protein-protein complex C ellular responses to extracellular signals depend on a complex array of protein-protein interactions, including signaling cascades within the cytoplasm involving different types of posttranslational modification, ultimately leading to the activation of factors that mediate transcription of the appropriate response genes. One of the best-understood signaling molecules is NF-κB, a family of inducible transcription factors that is present in almost all cell types in higher eukaryotes. Its activation through various stimuli elicits variable responses, leading to transcription of target genes necessary for the immune response, inflammation, cellular growth, differentiation, cell adhesion, and cell survival (1). In mammals, the NF-κB family comprises a set of homo-or heterodimers of five monomer units, termed RelA (also known as p65), RelB, c-Rel, p50, and p52. The most abundant and bestknown NF-κB is the heterodimer of RelA and p50, present in the cytoplasm of resting cells in complex with its inhibitor IκBα, which masks the nuclear localization signal (NLS) of RelA. Upon receipt of a stimulus, a signaling cascade results in the phosphorylation, ubiquitination, and degradation of the bound IκBα; the RelA NLS is released; and the NF-κB enters the nucleus. Signal-responsive genes are regulated by the binding of NF-κB to the appropriate promoter. The activated NF-κB response is terminated through various mechanisms, including resynthesis of IκBα; the corresponding gene, nfkbia, is a strongly induced target gene of NF-κB. Newly synthesized IκBα enters the nucleus, recombines with the DNA-bound NF-κB, and the complex reverts to the resting state in the cytoplasm. The newly synthesized IκBα acts as an important component of the negative feedback loop that is one of the mechanisms for termination of NF-κB activation (2).A number of studies have illuminated the structural basis for the protein-protein interactions that NF-κB undergoes during the activation process. The X-ray crystal structure of the complex of RelA-p50 with a cognate DNA ...

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“…Murine N-terminal hexahistidine-RelA 19-321 /p50 39-350 heterodimer (NFκB) was coexpressed as described previously (20) and was purified by nickel affinity chromatography, cation exchange chromatography (Mono S; GE Healthcare), and size-exclusion chromatography (Superdex 200; GE Healthcare). Murine dimerization domain RelA with an N-terminal cysteine and dimerization domain p50 248-350 were expressed, purified, and prepared for surface plasmon resonance (SPR) as described (25,26,29,30). Immediately before the experiments, IκBα was purified by size-exclusion chromatography (Superdex 75; GE Healthcare), and NFκB and NFκB-IκBα complexes were purified by size-exclusion chromatography (Superdex 200) in 25 mM Tris (pH 7.5), 150 mM NaCl, 1 mM DTT, and 0.5 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

“…The mechanism of this process necessitates the formation of a ternary complex, which was extremely transient under stopped-flow conditions but could be observed under the high-concentration conditions of NMR experiments (11)(12)(13). Using coarse-grained simulations, we showed that at least part of the driving force for molecular stripping likely comes from electrostatic repulsion between the negatively charged IκBα PEST sequence and DNA (25). Here, we undertook a comprehensive experimental characterization of the effects of PEST neutralization on the kinetics of molecular stripping, on the structure of the transient ternary complex, and on the function of IκBα.…”

Section: Stripping-impaired Iκbα Is Deficient In Regulating Nfκb In Kmentioning

confidence: 95%

Functional importance of stripping in NFκB signaling revealed by a stripping-impaired IκBα mutant

Dembinski

Wismer

Vargas

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Stress-response transcription factors such as NFκB turn on hundreds of genes and must have a mechanism for rapid cessation of transcriptional activation. We recently showed that the inhibitor of NFκB signaling, IκBα, dramatically accelerates the dissociation of NFκB from transcription sites, a process we have called "stripping." To test the role of the IκBα C-terminal PEST (rich in proline, glutamic acid, serine, and threonine residues) sequence in NFκB stripping, a mutant IκBα was generated in which five acidic PEST residues were mutated to their neutral analogs. This IκBα(5xPEST) mutant was impaired in stripping NFκB from DNA and formed a more stable intermediate ternary complex than that formed from IκBα(WT) because DNA dissociated more slowly. NMR and amide hydrogen-deuterium exchange mass spectrometry showed that the IκBα(5xPEST) appears to be "caught in the act of stripping" because it is not yet completely in the folded and NFκB-bound state. When the mutant was introduced into cells, the rate of postinduction IκBα-mediated export of NFκB from the nucleus decreased markedly.transcription factor | binding kinetics | intrinsically disordered proteins | nuclear export | hydrogen-deuterium exchange S tress-response transcription factors turn on hundreds of genes, and their regulation requires robust activation as well as rapid and complete cessation of the ensuing response. A good example is the NFκB family of transcription factors, which responds to a large number of extracellular stress stimuli, including factors controlling inflammation and the immune response (1-3). Aberrant regulation of NFκB results in numerous disease states, including cancer (1, 4). The IκB family of inhibitors keeps NFκB in the cytoplasm (in the "off" state) (5). IκBα is the main temporally regulated IκB. When a stress signal is received, IκBα is degraded rapidly, releasing NFκB, which enters the nucleus, binds to κB DNA sites, and up-regulates gene expression (Fig. 1A). In a classic negative feedback loop, the promoter upstream of the IκBα gene is strongly up-regulated by NFκB. We previously showed that in vitro IκBα rapidly accelerates the dissociation of NFκB from many different DNA sequences containing the κB motif in a folding-upon-binding event (6, 7). Thus, removal of NFκB(RelA/p50) from its target sites is kinetically determined, a process we call "molecular stripping" (8). The kinetic control of transcription factor-DNA interactions represents a paradigm shift because these interactions typically are described with equilibrium-binding models (9, 10) and thus would require the formulation of novel models based on stochastic rates.For IκBα to strip NFκB from DNA, a ternary NFκB-DNA-IκBα complex must form at least transiently. A very transient NFκB-DNA-IκBα complex was indeed observed in stopped-flow fluorescence experiments (11). At high concentrations, signals corresponding to a ternary NFκB-DNA-IκBα complex were also observed by NMR (12, 13). Together the stopped-flow and NMR data showed that IκBα binds to the NFκB-DNA comple...

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Molecular stripping in the NFκB/IκB/DNA genetic regulatory network

Cited by 11 publications

References 26 publications

N-methyl pyrrolidone (NMP) ameliorates the hypoxia-reduced osteoblast differentiation via inhibiting the NF-κB signaling

N-methyl pyrrolidone (NMP) ameliorates the hypoxia-reduced osteoblast differentiation via inhibiting the NF-κB signaling

Structural characterization of the ternary complex that mediates termination of NF-κB signaling by IκBα

Functional importance of stripping in NFκB signaling revealed by a stripping-impaired IκBα mutant

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